The invention relates to the field of gene expression and gene therapy, and to novel vectors for these uses. In particular, the invention relates to the development and use of an artificial or synthetic chromosome as a vector for gene expression and gene therapy, especially in humans. The invention enables the controlled construction of stable synthetic or artificial chromosomes from isolated purified DNA. With this DNA, a functional chromosome is formed in a cell and maintained as an extrachromosomal element. The artificial chromosome performs the essential chromosomal functions of naturally-occurring chromosomes so as to permit the chromosome to function as an effective vector for gene therapy when therapeutic DNA is included in the chromosome.
The genetic manipulation of cells aimed at correcting inherited or acquired disease is referred to as gene therapy. Until now, most clinical studies in this field have focused on the use of viral gene therapy vectors. Based on the results of these studies, it is becoming clear that current viral gene therapy vectors have severe clinical limitations. These include immunogenicity, cytopathicity, inconsistent gene expression, and limitations on the size of the therapeutic gene. For these reasons, much attention has been recently focused on the use of non-viral gene therapy vectors.
In particular, synthetic mammalian chromosomes would be useful vectors for facilitating a variety of genetic manipulations to living cells. The advantages of synthetic mammalian chromosomes include high mitotic stability, consistent and regulated gene expression, high cloning capacity, and non-immunogenicity.
Artificial chromosomes were first constructed in S. cerevisiae in 1983 (Murray et al., Nature 305:189-193 (1983), and in S. pombe in 1989 (Hahnenberger et al., Proc. Natl. Acad Sci. USA 86:577-581 (1989). For many reasons, however, it has not been obvious whether similar vectors could be made in mammalian cells.
First, multicellular organisms (and thus the progenitors of mammalian cells) diverged from yeast over 1 billion years ago. Although there are similarities among living organisms, in general, the similarities among two organisms are inversely related to the extent of their evolutionary divergence. Clearly, yeast, a unicellular organism, is radically different biologically from a complex multicellular vertebrate.
Second, yeast chromosomes are several orders of magnitude smaller than mammalian chromosomes. In S. cerevisiae and S. pombe, the chromosomes are 0.2 to 2 megabases and 3.5-5.5 megabases in length, respectively. In contrast, mammalian chromosomes range in size from approximately 50 megabases to 250 megabases. Since there is a significant difference in size, it is not clear, a priori, whether constructs comparable to yeast artificial chromosomes can be constructed and transfected into mammalian cells.
Third, yeast chromosomes are less condensed than mammalian chromosomes. This implies that mammalian chromosomes rely on more complex chromatin interactions in order to achieve this higher level of structure. The complex structure (both DNA structure and higher order chromatin structure) of mammalian chromosomes calls into question whether artificial chromosomes can be created in mammalian cells.
Fourth, yeast centromeres are far less complex than mammalian centromeres. In S. cerevisiae, for example, the centromere is made up of a 125 bp sequence. In S. pombe, the centromere consists of approximately 2 to 3 copies of a 14 kb sequence element and an inverted repeat separated by a core region (xcx9c7 kb). In contrast, human centromeres are made up of several hundred kilobases to several megabases of highly repetitive alpha satellite DNA. Furthermore, in mammalian centromeres, there is no evidence for a central core region or inverted repeats such as those found in S. pombe. Thus, unlike yeast centromeres, mammalian centromeres are extremely large and repetitive.
Fifth, yeast centromeres have far fewer spindle attachments than mammalian centromeres (Bloom, Cell 73:621-624 (1993)). S. cerevisiae, for example, has a single microtubule attached to the centromere. In S. Pombe, there are 2-4 microtubules attached per centromere. In humans, on the other hand, there are several dozen microtubules attached to the centromere of each chromosome (Bloom, Cell 73:621-624 (1993)). This further illustrates the complexity of mammalian centromeres compared to yeast centromeres.
Together, these differences are significant, and do not suggest that a result in yeast can be reasonably expected to be transferable to mammals.
Normal mammalian chromosomes are comprised of a continuous linear strand of DNA ranging in size from approximately 50 to 250 megabases. In order for these genetic units to be faithfully replicated and segregated at each cell division, it is believed that they must contain at least three types of functional elements: telomeres, origins of replication, and centromeres.
Telomeres in mammals are composed of the repeating sequence (TTAGGG)n and are thought to be necessary for replication and stabilization of the chromosome ends. Origins of replication are necessary for the efficient and controlled replication of the chromosome DNA during S phase of the cell cycle. Although mammalian origins of replication have not been well-characterized at the sequence level, it is believed that they are relatively abundant in mammalian DNA. Finally, centromeres are necessary for the segregation of individual chromatids to the two daughter cells during mitosis to ensure that each daughter cell receives one, and only one, copy of each chromosome. Like origins of replication, centromeres have not been defined at the sequence level. Alpha satellite DNA may be an important centromeric component (Haaf et al., Cell 70:681-696 (1992); Larin et al., Hum. Mol. Genet. 3:689-695 (1994); Willard, Trends in Genet. 6:410-415 (1990)). But there are cases of mitotically stable abnormal chromosome derivatives that apparently lack alpha satellite DNA (Callen et al., Am. J. Med. Genet. 43:709-715 (1992); Crolla et al., J. Med. Genet. 29:699-703 (1992); Voullaire et al., Am. J. Hum. Genet. 52:1153-1163 (1993); Blennow et al., Am. J. Hum. Genet. 54:877-853 (1994); Ohashi et al., Am. J. Hum. Genet. 55:1202-1208 (1994)). Thus, at this time, the composition of the mammalian centromere remains poorly understood.
While others have claimed to have produced xe2x80x9cartificialxe2x80x9d chromosomes in mammalian cells, no one has ever produced an artificial chromosome that contains only exogenous DNA. In each of these previous cases, the investigators either modified an existing chromosome to make it smaller (the xe2x80x9cpare-downxe2x80x9d approach) or they integrated exogenous DNA into an existing chromosome which then broke to produce a chromosome fragment containing endogenous sequences from the preexisting chromosome (the xe2x80x9cfragmentationxe2x80x9d approach). In the present invention, exogenous DNA sequences are introduced into human cells and form stable synthetic chromosomes without integration into endogenous chromosomes.
Among the pare-down approaches, three specific strategies have been used: (1) telomere directed truncation via illegitimate recombination (Barnett, M. A. et al., Nucleic Acids Res. 21:27-36 (1993); Farr, C. J. et al., EMBO J. 14:5444-54 (1995)) (2) alpha satellite targeted telomere insertion/truncation via homologous recombination (Brown, K. E. et al., Hum Mol. Genet. 3:1227-37 (1994)) (3) formation/breakage of dicentric chromosomes (Hadlaczlky, G., Mammalian Artificial Chromosomes, U.S. Pat. No. 5,288,625 (1994)).
Barnett et al. (Nucleic Acids Res. 21:27-36 (1993)), Farr et al. (EMBO J. 14:5444-54 (1995)), and Brown et al. (Hum Mol. Genet. 3:1227-37 (1994)) describe methods for fragmenting endogenous chromosomes by transfecting telomeric DNA and a selectable marker into mammalian cells. In each case, a truncated chromosome was created that was smaller than the original chromosome. The resulting truncated chromosomes contained large amounts of endogenous chromosome sequence, including the endogenous centromere. Thus, these chromosomes were not formed de novo.
Hadlaczky (Mammalian Artificial Chromosomes, U.S. Pat. No. 5,288,625 (1994)) describes a cell-line that can be use to propagate a chromosome that was formed as a result of a dicentric chromosome breakage event. All of the sequences, with the exception of a selectable marker were derived from the original, fully functional dicentric chromosome. Thus, these so called xe2x80x9cartificialxe2x80x9d chromosomes were not created de novo.
Among the xe2x80x9cfragmentationxe2x80x9d approaches, Haaf et al. (Cell 70:681-696 (1992)) and Praznovszky et al. (Proc. Natl. Acad Sci. USA 88:11042-11046 (1991)) describe methods for producing chromosome fragments by integrating transfected DNA into endogenous chromosomes. Following transfection, the integrated DNA sequences become amplified (increase in copy number), and in some clones, a portion of the endogenous chromosome breaks off to produce a fragment that exists extrachromosomally. In both references, integrated transfected DNA can be found extensively on the endogenous chromosome and the extrachromosomal fragment.
In the experiments by Haaf et al. (Cell 70:681-696 (1992)), human alpha satellite DNA and the neomycin resistance gene were co-transfected into African Green Monkey cells. No other exogenous DNA was included in any of the transfections. In every transfection clone, DNA was found to be integrated into the endogenous chromosomes. In one clone, which was also found to contain an extrachromosomal fragment, the transfected alpha satellite DNA had amplified extensively following integration. The authors conclude, based on Southern blot and Fluorescence In-Situ Hybridization, that African Green Monkey sequences co-amplified with the transfected DNA and were interspersed among the alpha satellite DNA. In further characterization of the chromosomes that contained amplified alpha satellite, it was found that xe2x80x9cthe number, size, and chromosomal location (telomeric, interstitial, or centromeric) of the transfected chromosome regions varied from cell to cell within the population of line 3-31 cells, suggesting instability of the transfected sequences.xe2x80x9d Finally, analysis of the mitotic behavior of the chromosomes containing amplified alpha satellite DNA revealed a high incidence of anaphase bridges, suggesting that the chromosomes were dicentric (or multicentric). Thus, the high degree of observed structural instability in conjunction with the high incidence of anaphase bridge structures is consistent with the idea that the chromosome fragment resulted from an integration/amplification/breakage event. Finally, it is also worth noting that in clones that contained integrated, unamplified alpha satellite DNA, no extrachromosomal fragments were observed, further suggesting that amplification is important for the chromosome fragmentation process in this method.
Praznovszky et al. (Proc. Natl. Acad Sci. USA 88:11042-11046 (1991)) produced chromosome fragments by integrating a piece of non-centromeric human DNA (later shown to map to human chromosome 9 qter by McGill et al. (Hum. Mol. Genet. 1:749-751 (1992)) and Cooper et al. (Hum. Mol. Genet. 1:753-754 (1992)) into an endogenous chromosome. Like the Haaf experiment, the integrated transfected DNA amplified extensively, and was found to be interspersed with mouse genomic sequences. The authors suggest that the integration/amplification of the transfected DNA resulted in the formation of a dicentric chromosome that then subsequently broke to produce chromosome fragments. Analysis of the chromosome fragments shows unambiguously that the chromosome fragments were derived from the mouse chromosome containing the integrated amplified DNA.
There are a number of important similarities between the experiments by Haaf et. al. and Praznovszky et. al. First, both show that the transfected DNA integrated into endogenous chromosomes. Second, both show that following integration, the transfected DNA amplified extensively. Third, endogenous DNA (untransfected chromosomal sequences from the recipient cell ) was found to be interspersed throughout the amplified sequences. Fourth, the endogenous chromosomes containing the amplified transfected sequences stained with CREST antisera. Fifth, the endogenous chromosomes containing the amplified transfected sequences behaved similarly to dicentric chromosomes during mitosis. Finally, the endogenous chromosomes containing the amplified transfected sequences displayed structural instability. Thus, the large number of important similarities and the demonstrated chromosomal fragmentation by Praznovszky et. al. indicate a chromosome integration/amplification/breakage mechanism in both of these experiments.
Further evidence that transfection and integration of alpha satellite DNA into mammalian chromosomes is not sufficient to create extrachromosomal fragments in the absence of amplification was obtained by Larin et. al. (Hum. Mol. Genet. 3:689-95 (1994)). In these experiments, alpha satellite DNA linked to a selectable marker was transfected into human cells. In every drug-resistant clone, the alpha satellite DNA was integrated into an endogenous chromosome. While these integrations formed centromere-like structures (i.e. primary constrictions, CREST antisera staining, and lagging chromosomes during anaphase), no extrachromosomal fragments were observed in any clone. Since these experiments failed to provide clones with chromosomes containing the transfected alpha satellite DNA and not an endogenous centromere, there is no reliable method to determine whether the centromere-like structures that formed are capable of facilitating chromosome segregation.
Since each of the xe2x80x9cpared-downxe2x80x9d chromosomes was created from a pre-existing chromosome and since each of the xe2x80x9cfragmentationxe2x80x9d chromosomes was created by integrating DNA into pre-existing chromosomes, these references do not provide guidance about how to create chromosomes de novo from transfected naked DNA.
Furthermore, these chromosomes and the approaches used to make them have severe limitations as gene therapy vectors for several reasons. First, the methods used to make them can only be used to create the chromosomes in cell culture. Since the breakage events are either extremely rare and/or produce chromosomes with unpredictable structure, these methods are not compatible with direct use in patients"" cells. Additionally, the instability of the amplified sequences in the fragmentation approach is inconsistent with use in patients due to the risks of genomic rearrangements that, in turn, may lead to cellular transformation and cancer.
It would be highly desirable, therefore, if there were a prefabricated chromosome vector with defined structure that could be introduced directly into patient""s cells, especially a vector that did not depend upon integration into endogenous chromosomes or subsequent amplification, and where the structure of the construct in the cell is substantially identical to its structure prior to transfection.
Second, pared-down chromosomes and chromosome fragments are composed of undefined endogenous sequences and provide no guidance for identifying sequences that are functionally important.
It would be highly desirable, therefore, to provide vectors composed of defined sequences and the methods to produce these defined synthetic chromosomes that allow other functionally important sequences to be rapidly identified.
Third, the chromosomes produced by the pare-down and fragmentation approaches can not be substantially purified using currently available techniques. Thus, it is difficult to deliver these pared-down chromosomes to mammalian cells without delivering other mammalian chromosomes.
It would be highly desirable, therefore, to provide substantially purified genetically engineered DNA that can be introduced into a cell and form a functional chromosome.
Fourth, since these pared-down chromosomes and chromosome fragments have never been isolated as naked DNA and reintroduced into a cell, up to the present, it was never clear whether any exogenous DNA could be introduced into a cell to produce a functional chromosome de novo (without integrating into the host chromosomes first).
It would be highly desirable, therefore, to provide artificial mammalian chromosomes that are created de novo by introducing purified DNA into a mammalian cell.
Finally, it is very difficult to add new DNA sequences (e.g. therapeutic genes) to the pared-down chromosomes and chromosome fragments.
It would be highly desirable, therefore, to provide vectors created in vitro, where placing new DNA sequences onto the vectors is straight-forward and efficient.
Sun et. al. (Nature Genetics 8:3341 (1994)) describe a viral-based vector system designed for use in human cells. The vector is described as a xe2x80x9chuman artificial episomal chromosome.xe2x80x9d However, the vector relies on the presence of EBNA-1, a toxic and immunogenic viral protein. Further, the vector relies on a viral origin of replication and not on a natural mammalian chromosomal replication origin. Further, the xe2x80x9cchromosomexe2x80x9d does not contain functional centromeric or telomeric DNA, and does not form a functional kinetochore during mitosis. As a result, such a vector does not segregate in a controlled manner. Finally, the vector is present in the cell at an elevated copy number that ranges from 50 to 100 copies per cell, unlike endogenous chromosomes. Based on these criteria for defining mammalian chromosomes, this vector cannot be properly designated a xe2x80x9chuman artificial chromosomexe2x80x9d because it has different properties and functions by unrelated mechanisms.
Thus, there is still a clear need for a wholly synthetic or artificial chromosome made from DNA that can be manipulated in vitro and, upon transfection into cells, will adopt a functional chromosome structure and will direct gene expression in a controlled manner.
In contrast to the cited art, several embodiments of the current invention describe a prefabricated chromosome vector with defined structure and composition that can be introduced directly into patients"" cells. Since the vector described in this invention does not depend upon integration into endogenous chromosomes or subsequent amplification, the structure of the construct in the cell is substantially identical to its structure prior to transfection.
In contrast to the cited art, the vectors described in this present invention are composed of defined sequences. Furthermore, the methods used to produce these synthetic chromosomes allow other functionally important sequences to be rapidly identified.
In contrast to the cited art, with the present invention, the inventors demonstrate for the first time that artificial mammalian chromosomes can be created de novo by introducing purified DNA into a mammalian cell.
In contrast to the cited art, since the vectors described in the present invention are created in vitro, placing new DNA sequences onto the vector is straight-forward and efficient.
The inventors have developed methods for producing large quantities of purified intact alpha satellite arrays of up to 736 kb in length. By transfecting these arrays into human cells along with telomeric DNA and human genomic DNA sequences, several wholly synthetic human chromosomes that exhibit a high degree of mitotic stability in the absence of selection have been produced.
Unlike previous approaches whereby attempts were made to produce an artificial mammalian chromosome, this approach does not rely on the modification of existing endogenous chromosomes. Furthermore, it does not produce multiple integration events within the endogenous chromosomes. These chromosomes were formed and maintained extrachromosomally, so integration into an endogenous chromosome is avoided.
The relatively high frequency of synthetic chromosome formation and the lack of other genomic rearrangements associated with the chromosome formation, allows the synthetic chromosomes made by the inventors to be used as effective vectors for heterologous gene expression and gene therapy.
The invention is thus based on the inventors"" discovery that by means of isolated purified DNA alone, a synthetic or artificial chromosome is produced de novo (from purified DNA) in a cell and is produced and maintained as an extrachromosomal element. This chromosome retains the essential functions of a natural mammalian chromosome in that it is stably maintained as a non-integrated construct in dividing mammalian cells without selective pressure, just as naturally-occurring chromosomes are inherited. For a linear chromosome, this indicates centromeric, telomeric, and origin of replication functions.
The invention is thus directed to a synthetic or artificial mammalian chromosome. The chromosome is produced from isolated purified DNA. The isolated purified DNA is transfected into mammalian cells. Without integrating into an endogenous chromosome, it forms a functional chromosome. This chromosome is not derived from an endogenous naturally-occurring chromosome in situ. The starting material is isolated purified centromeric DNA and DNA that allows chromosome formation without integration. For linear chromosomes, telomeric DNA is included. In a preferred embodiment, the DNA that allows chromosome formation without integration is genomic DNA (from the naturally-occurring genome of an organism).
The artificial mammalian linear chromosome thus preferably essentially comprises centromeric, telomeric, and genomic DNA. In one embodiment, the artificial chromosome is a circular chromosome. In this case, telomeric DNA is absent since it is not necessary to replicate chromosome ends.
The genomic DNA is a subgenomic DNA fragment that is a restriction enzyme digestion fragment, a fragment produced by mechanical shearing of genomic DNA, or a synthetic fragment synthesized in vitro. The genomic DNA starting material (ie., that is transfected) can be a mixture of heterogeneous fragments (e.g., a restriction digest) or can be a cloned fragment or fragments (homogeneous).
Centromeric DNA comprises a DNA that directs or supports kinetechore formation and thereby enables proper chromosome segregation. Centromeric DNA at active, functional, centromeres is associated with CENP-E during mitosis, as demonstrated by immunofluorescence or immunoelectron microscopy. By xe2x80x9cassociatedxe2x80x9d is meant that the centromeric DNA and CENP-E co-localize by fluorescence in situ hybridization (FISH) and immunofluorescence.
Telomeric DNA comprises tandem repeats of TTAGGG that provide telomere function, i.e., replicate the ends of linear DNA molecules. Telomeric DNA is included as an optional component, to be used when linear chromosomes are desired. This is indicated herein by enclosing the terms xe2x80x9ctelomericxe2x80x9d/xe2x80x9ctelomerexe2x80x9d in parentheses.
Prior to transfection, the DNA can be naked, condensed with one or more DNA-condensing agents, or coated with one or more DNA-binding proteins.
The invention is also directed to an artificial mammalian chromosome produced by the process of introducing into a mammalian cell the isolated purified DNA fragments above. In a preferred embodiment the process uses DNA essentially comprising centromeric, telomeric, and genomic DNA.
The various fragments can be transfected separately or one or more can be ligated prior to transfection. Thus the centromeric (telomeric) and genomic DNAs are introduced separately (unligated) or one or more of the isolated purified DNAs are ligated to one another.
The invention is also directed to a mammalian cell containing and compositions comprising the artificial mammalian chromosome.
The invention is also directed to the isolated purified DNA described above, and which forms an artificial mammalian chromosome when introduced into a mammalian cell. In preferred embodiments, the isolated purified DNA essentially comprises centromeric, telomeric, and genomic DNA.
The invention is also directed to a mammalian cell containing and compositions comprising the purified DNA.
The invention is also directed to a vector or vectors containing the purified DNA.
The invention is also directed to a mammalian cell containing and compositions comprising the vector(s).
The invention is also directed to the isolated purified DNA described above produced by the process of combining one or more of the DNAs described above. In preferred embodiments, the DNA includes: (1) centromeric DNA, (2) telomeric DNA, (3) genomic DNA. The DNAs can be unligated or one or more can be ligated to one another.
The invention is also directed to a method for making an artificial mammalian chromosome by introducing into a mammalian cell the purified DNA described above.
The invention is also directed to a method for making DNA capable of forming an artificial chromosome, the method comprising combining in vitro the DNA described above.
The invention is also directed to a method for propagating an artificial chromosome in mammalian cells by introducing the purified DNA into a mammalian cell and allowing the chromosome to replicate.
In a preferred embodiment, the invention is also directed to methods for expressing a heterologous gene in a mammalian cell by expressing that gene from the artificial mammalian chromosome.
Thus, the invention is also directed to methods for providing a desired gene product by including a desired gene on the artificial chromosome such that the gene of interest is expressed. In preferred embodiments, the invention provides a method of gene therapy by including heterologous therapeutic DNA on the artificial mammalian chromosome, such that there is a therapeutic effect on the mammal containing the chromosome.
In a preferred embodiment of the invention, the centromeric DNA is alpha-satellite DNA.
In a preferred embodiment of the invention, the artificial mammalian chromosome is derived entirely from human DNA sequences and is functional in human cells.